PARP-1体外活性测定方法的建立及其应用

Establishment and Application of Enzymatic Assay for Poly(ADP-Ribose) Polymerase-1

建立PARP-1的体外活性测定方法并对84个具有潜在PARP抑制活性的化合物进行筛选。从Sf9昆虫细胞中提取酶液,以NAD+为底物,DNA为激活剂,建立PARP-1的活性测定方法;以高通量技术参数评价此方法,用已知PARP抑制剂进行验证,并以此方法进行PARP抑制剂的筛选。结果显示,确定的酶促反应体系为:12.5 nmol/L NAD+,15μg/m L DNA,6.25×10-4U PARP-1。评价Z'因子为0.76,S/B值为3.58。基于此模型筛出47个化合物在浓度7.4μmol/L时对PARP-1的抑制率达到60%以上。表明所建立的PARP-1活性测定方法,适用于PARP-1抑制剂的高通量筛选。

To establish an enzymatic assay for poly（ADP-ribose） polymerase-1 and screen PARP inhibitors from 84 potential compounds.Crude PARP-1 was extracted from Sf9 insect cells.With nicked DNA as activator,PARP-1 was used to catalyze the hydrolysis of the substrate NAD＋,the RFU value measured from the remaining NAD^＋was used to evaluate the activity of PARP-1 or efficiency of PARP inhibitors.A 96-microwell screening method was set up by optimizing the reaction system.The screening method was assessed with high throughput index,such as Z＇ factor,signal to back ground（S/B） and validated by well-known PARP inhibitors such as PHE,DPQ and AZD2281.84 compounds were assayed for PARP inhibition through the established method.The enzymatic reaction system was optimized as：12.5 nmol/L NAD^＋,15 μg/m L DNA,6.25 ×10^-4U PARP-1.The Z＇ factor is 0.76,S/B is 3.58 and the IC50 of PHE,DPQ and AZD2281 is 562.4 nmol/L,369.6 nmol/L and 6.8 nmol/L.There are 47 compounds showed 60% or more PARP-1 inhibition efficiency at the concentration of 7.4 μmol/L.An enzymatic assay of PARP-1 was successfully established.It can be effectively applied to high-throughput screening for PARP inhibitors.